Biomedical subjects

P Diel

Publications and source records attributed to P Diel.

25 records · Page 2Linked to original sources

PubMed1999

In vitro test systems for the evaluation of the estrogenic activity of natural products.

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. There is evidence that the consumption of some of these phytoestrogens may have beneficial effects but it also seems possible that others may act as endocrine disrupters. For this reason there is a need to characterise the estrogenic potency of these substances. In vitro test systems offer the possibility to screen compounds very efficiently. Routinely in use and widespread for the determination of estrogenicity are: (I) receptor binding assays, (II) cell-proliferation assays (E-screens), (III) reporter gene assays, and (IV) the analysis of the regulation of endogenous estrogen sensitive genes in cell lines. The basis of all these test systems are molecular mechanisms which are involved in the classical estrogen action. In addition, in the last years several test systems for the investigation of non-classical estrogenic effects have been established. An example for such an effect is the modulation of the expression of interleukin-6, a cytokine that appears to be a key molecule in the osteoporotic process, by estrogens. Summarising the advantages and the issues of all presented in vitro test systems, it seems to be evident that only the analysis of results obtained in a combination of several in vitro test systems may validly predict effects in vivo.

Estrogens, Non-Steroidal↗

PubMed1998

Effect of 17beta-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta.

1. Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17beta-oestradiol on cytokine-induced nitric oxide production by the inducible nitric oxide synthase (iNOS). 2. Treatment of the isolated aortic rings with interleukin-1beta (IL-1beta, 20 micro ml(-1)) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM-10 microM)-evoked contraction. 3. Cycloheximide (1 microM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL-1beta on the isolated aortic rings. 17Beta-oestradiol (1 nM-10 microM) and the partial oestrogen receptor agonist 4-OH-tamoxifen (1 nM-10 microM) produced concentration-dependent inhibition of IL-1beta-induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 microM). 4. Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL-1beta-induced and 17beta-oestradiol-treated rings. Western blot analysis of rat aorta homogenates revealed that 17beta-oestradiol treatment resulted in a reduction in IL-1beta-induced iNOS protein level. 5. Incubation with tumour necrosis factor alpha (TNF alpha, 1 ng ml(-1)) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL-1beta. The effects of TNF alpha were also inhibited by co-incubation of the rings with 17beta-oestradiol and 4-OH-tamoxifen (1 microM). 6. The anti-transforming growth factor-beta1 (TGF-beta1) antibody, which inhibited TGF-beta1-induced suppression of nitrite production from IL-1beta-treated vascular rings, did not affect the inhibitory action of 17beta-oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF-beta1. 7. These results show that the ovarian sex steroid, 17beta-oestradiol is a modulator of cytokine-induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

Animals↗

PubMed1996

Sexual dimorphism of steroid hormone receptor messenger ribonucleic acid expression and hormonal regulation in rat vascular tissue.

Evidence has accumulated suggesting that steroid hormones have a direct effect on the vascular system. Of special interest is the protective effect of estrogens against cardiovascular diseases. One of the aims of the present study was to investigate the messenger RNA (mRNA) expression of the five classic steroid hormone receptors in the great vessels of the rat (aorta, vena cava, and vena portae) to provide a basis to analyze direct steroid effects on vascular tissue. By applying reverse transcription-PCR we compared the expressions of the steroid hormone receptor mRNAs in the respective vessels of male and female rats. Sex differences in the mRNA levels of the mineralocorticoid (MR), estrogen (ER), and progesterone (PR) receptors were found in venous vessels, but not in the aorta. Focussing on the vena cava in the female rat, we investigated whether the ER is active in the vasculature by analyzing regulation of the PR gene. This gene is known to be regulated by estrogens in classic target organs. PR mRNA expression in venous vessels of female rats decreased after ovariectomy. This effect was reversed by chronic sc treatment with estradiol (E2; 1 microgram/animal day). Progester-one (10 mg/animal day, sc) partly inhibited the effect of E2. Besides E2, the partial agonist tamoxifen stimulated PR mRNA expression in ovariectomized rats, whereas the pure antiestrogen ZM 182780 remained inactive in this experiment. Both E2 and tamoxifen caused an autologous down-regulation of ER mRNA. In conclusion, our study demonstrates for the first time that not only the aorta, but also the venous vessels, represented by the vena cava and the portal vein of the rat, are targets for steroid hormones. The ER in vascular tissue is functionally active and mediates direct modulatory effects of estrogens on gene expression in vascular cells.

Animals↗

PubMed1996

Multiple signal transduction pathways regulate clusterin (gp 80) gene expression in MDCK cells.

Clusterin (gp 80, apolipoprotein J, TRPM-2) is a widely expressed multifunctional glycoprotein. Its demonstrated and proposed functions include the transport of lipids and membrane fragments, the inhibition of the cytolytic action of the terminal complement complex and the modulation of cell-cell interactions. The expression of the gene is enhanced during tissue injury and remodelling and by hormone-withdrawal-induced apoptosis of prostate and mammary cells. We show here that, in the kidney-derived epithelial cell line MDCK, clusterin mRNA is repressed by glucocorticoids and by progesterone. Treatment with epidermal growth factor also represses clusterin gene expression in MDCK cells. Incubation with 12-O-tetradecanoyl-phorbol-13-acetate, which activates protein kinase C (PKC), induces clusterin mRNA, while chelerythrine, an inhibitor of PKC, represses clusterin gene expression, suggesting that the clusterin gene responds to signalling pathways involving PKC. These results open up the possibility of studying the complex regulation of the clusterin gene by multiple signal transduction pathways within a single cell type, and most importantly, of characterizing interactions between the individual signal transduction cascades.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Identification of estrogen regulated genes in Fe33 rat hepatoma cells by differential display polymerase chain reaction and their hormonal regulation in rat liver and uterus.

We applied the differential display RT-PCR (ddRT-PCR) technology to identify estrogen-regulated hepatic genes in the estrogen receptor expressing rat hepatoma cell line Fe33. Three genes of known sequences were detected by the ddRT-PCR approach: IGF binding protein-1 (IGFBP-1), vitamin D-dependent calcium-binding protein (CaBP9k) and major acute phase protein (MAP). Effects of ethinyl estradiol on the mRNA levels of these genes were confirmed by "Northern-blot" analysis. If given in combination with dexamethasone and glucagon, ethinyl estradiol caused 40-, 15- and 11-fold increases in the mRNA steady state level of IGFBP-1, CaBP9k and MAP, respectively, in Fe33 cells 24 h after addition of hormone. Besides ethinyl estradiol, the partial estrogen agonist OH-tamoxifen caused dose dependent effects on expression of MAP and IGFBP-1. Estrogen regulation of the respective genes and the modulatory effects of progesterone (10 mg/animal/day) were studied in ovariectomized rats treated subcutaneously for 14 days with 1 microgram/animal/day estradiol. "Northern-blot" analysis of liver RNA revealed a 6-fold stimulation of IGFBP-1 mRNA levels in estradiol-treated compared to vehicle-treated rats and a weak but detectable increase of MAP mRNA steady state level (1.6-fold) upon estradiol administration. No effect of estradiol treatment could be monitored for CaBP9k in rat liver. Modulatory effects of progesterone on estradiol-stimulated expression in the liver could be monitored for IGFBP-1 only. In an extension of our investigation on the expression of the three genes in rat liver, we determined their expression and hormonal regulation in the uterus of the same animals. In the uterus, estradiol caused an increase in CaBP9k mRNA. In contrast, IGFBP-1 mRNA levels increased dramatically upon progesterone administration, whereas no effect of estradiol treatment could be detected. MAP mRNA levels increased only after coadministration of estradiol and progesterone. In conclusion, the ddRT-PCR proved to be a powerful method to identify estrogen-regulated genes. The study on the hormonal regulation of three genes stimulated by estrogen in Fe33 cells revealed similarities and differences in their regulation in vivo and in vitro.

Acute-Phase Proteins↗

PubMed1991

Molecular cloning of gp 80, a glycoprotein complex secreted by kidney cells in vitro and in vivo. A link to the reproductive system and to the complement cascade.

cDNA clones coding for the gp 80 heterodimeric glycoprotein complex secreted constitutively at the apical surface of Madin-Darby canine kidney (MDCK) cells have been isolated from MDCK cDNA libraries in lambda gt11 and lambda gt10. The cloned sequences encode a polypeptide chain of 445 amino acids. The deduced amino acid sequence of the gp 80 protein reveals 80% homology to rat SGP-2, a major secretory protein of the testes epithelium and 83% homology to SP-40,40, a human complement-associated protein. SGP-2 and SP-40,40 have been proposed to be serum and seminal forms of the same protein. The sequence homology as well as the results of Southern and Northern blot analyses and immunological studies suggest that gp 80 is the canine homolog of the rat SGP-2 and the human SP-40,40. The protein is expressed in the embryonic kidney already early during organogenesis. In the adult kidney the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of a pure antiestrogen and progesterone on estrogen-mediated alterations of blood flow and progesterone receptor expression in the aorta of ovariectomized rabbits.

There is ample evidence from epidemiological studies that estrogen-replacement therapy protects postmenopausal women against cardiovascular disease. One explanation for this beneficial effect could be the improvement of blood flow under estrogen therapy. By using ultrasound and Doppler color flow mapping we demonstrated in the aorta of ovariectomized rabbits a significant dose-dependent increase in blood flow after treatment with 17beta-estradiol. An increase in blood flow was already observed within 1 h of estradiol treatment and lasted until the end of a 14-day treatment phase. Progesterone did not attenuate the effects of 17beta-estradiol on aortic blood flow. The pure estrogen receptor antagonist ZM 182780, however, dose-dependently reversed the effect of 17beta-estradiol on blood flow after the 14-day treatment phase, but was not able to antagonize the rapid 17beta-estradiol effect on blood flow after 1 h. After killing the animals mRNA and protein expression of the progesterone receptor (PR), a known estrogen-responsive gene in classic target organs, were examined. Analogous to the blood flow results the PR mRNA level increased dose-dependently after 17beta-estradiol treatment, whereas ZM 182780 was able to reverse this effect. Immunohistochemical localization of PR in the aortic wall revealed an increase in immunoreactivity in fibroblasts of the adventitia after 17beta-estradiol treatment. ZM 182780, and to a lesser degree progesterone, reversed the 17beta-estradiol-induced increase in PR immunoreactivity. PR immunoreactivity was further detected in endothelial and smooth muscle cells, but the various hormonal treatments had no discernible effect on the PR mRNA level in these cellular compartments. Our findings in the aorta of OVX rabbits suggest that (a) 17beta-estradiol exhibits a rapid effect on arterial tone, (b) the pure estrogen receptor antagonist ZM 182780 inhibits the 17beta-estradiol effect on blood flow and PR mRNA and (c) progesterone does not attenuate the beneficial effect of estrogens on arterial tone.

Animals↗